Week of 6/28

From 2010.igem.org

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 +
==June 28==
 +
 +
* Retransform:
 +
  -R0061
 +
  -R0062
 +
  -R0063
 +
  -K091146
 +
 +
*Inventory
 +
-Good Sequencing:
 +
    o Backbone - C3RFP
 +
    o pLux    - K091146
 +
    o Lysis    - K112022
 +
    o pLux    - R0063
 +
    o pLux    - R1062
 +
    o CI+RBS  - K081007
 +
    o Cons.Prom- J23116
 +
    o Cons.Prom- J23108
 +
    o Pigment  - K274100
 +
    o LuxI    - C0261
 +
    o Lysis    - K112808
 +
    o pLac    - R0010
 +
    o LacI+RBS - J24679
 +
 +
*To grow:
 +
    - K091146
 +
    - R0063
 +
    - J23108
 +
    - J23116
 +
 +
*Electrocompetent cells were made.
 +
 +
 +
==June 29==
 +
 +
Gel Order:
 +
    - J23108
 +
    - J23108 (Digest 2)
 +
    - R0063 
 +
    - R0063  (Digest 2)
 +
    - K081007
 +
    - K081007(Digest 2)
 +
    - J23116
 +
    - J23116 (Digest 2)
 +
 +
<center>[[Image:6-29-10.jpg]]</center>
<center>[[Image:6-29-10.jpg]]</center>
 +
 +
 +
*Gel extract was done using Digest number 1. In this gel we ran the same part twice but from two different digests. Digest 1 was a 6 hour digest in the 37 degree Celsius water bath while Digest 2 was a 1 hour digest in the same water bath. As you can see, the results do not differ all that noticeably. That being established we went back to doing 1 hour digests.
 +
 +
*The bands that were cut out are provided below:
 +
    - J23108
 +
    - R0063
 +
    - J23116
 +
    - K081007
 +
 +
All these bands were purified.
 +
 +
==June 30==
 +
 +
 +
*On this gel, we ran the Colony PCR and the digests. The order is as follows:
 +
 +
*Colony PCR:
 +
  - Ladder
 +
  - A-J
 +
 +
*Digests:
 +
  - Ladder
 +
  - K091107 (digest 2)
 +
  - K091107
 +
  - K091146 (digest 2)
 +
  - K091146
 +
  - J23116+J37033 (digest 2)
 +
  - J23116+J37033
 +
  - R0010 (digest 2)
 +
  - R0010
<center>[[Image:6-30-10.jpg]]</center>
<center>[[Image:6-30-10.jpg]]</center>
 +
*The bands that were cut are marked with a square around them in the picture above. They were also then purified.
 +
* 3 bands that were cut:
 +
    - K091107
 +
    - K091146
 +
    - R0010
 +
 +
 +
 +
*A second gel was run the same day. The order is provided below:
 +
  - Ladder
 +
  - B1002
 +
  - Lux R 2
 +
  - C0261+J24679
<center>[[Image:6-30-10 001.jpg]]</center>
<center>[[Image:6-30-10 001.jpg]]</center>
 +
 +
*As you can see, the only 2 bands that were cut were B1002 and C0261+J24679
 +
 +
 +
*Grow for tomorrow:
 +
  - K274100
 +
  - K112022
 +
 +
==July 1==
 +
 +
 +
*Flasks minipreped:
 +
    - B1006
 +
    - K274100
 +
    - J23100
 +
 +
*Digests:
 +
    - B1006 (EX)
 +
    - K274100 (XP)
 +
    - J23100 (SP)
 +
 +
*Sent to the sequencing center:
 +
  1) B1002
 +
  2) B1006
 +
  3) S01414
 +
  4) J23100
 +
  5) C0261+J24679
 +
 +
 +
*Gel order:
 +
  - Ladder
 +
  - B1006
 +
  - K274100
 +
  - J23100
 +
 +
<center>[[Image:7-1-10.jpg]]</center>
 +
 +
 +
*All the bands appeared where we wanted them to so we cut out all three bands and then purified them.
 +
 +
 +
 +
*Ligations:
 +
{|
 +
|Vector||Inserts
 +
|-
 +
|1)J23100||K274100
 +
|-
 +
|2)J23100||S01414
 +
|-
 +
|3)R0063||a)K274100<br />b)C0261 + J24679
 +
|-
 +
|4)K091146||a)K274100<br />b)C0261 + J24679
 +
|-
 +
|5)K091107||a)K274100<br />b)C0261 + J24679
 +
|-
 +
|6)R0062||a)K274100<br />b)C0261 + J24679
 +
|-
 +
|7)R0010||K081007
 +
|}
 +
 +
*Numbers 3-6 were each matched up with a) K274100 and b) C0261 + J24679. The rest of the vectors were matched with the insert directly to the right in the table.
 +
 +
 +
*Tomorrow:
 +
- Develop primers to figure out if K274100 and K112808 are correct.
 +
 +
==July 2==
 +
 +
 +
*Colony PCRs run on gel.The order is as follows:
 +
    - Ladder
 +
    - 1A, 1B, 1C
 +
    - 2A, 2B, 2C
 +
    - 3A, 3B, 3C
 +
 +
    - Ladder
 +
    - 4A, 4B, 4C
 +
    - 5A, 5B, 5C
 +
    - 6A, 6B, 6C
 +
    - 7A, 7B, 7C
 +
 +
<center>[[Image:7-2-10.jpg]]</center>
 +
 +
 +
Unfortunately, we accidentally put ladder in all the Colony products. The picture is provided above. We could not use this information in any way other than to learn from our mistake.
-
<center>[[Image:6-1-10.jpg]]</center>
 
-
<center>[[Image:6-2-10.jpg]]</center>
+
[https://2010.igem.org/Team:Penn_State/Notebook Return to Notebook]

Latest revision as of 15:42, 5 August 2010

Contents

June 28

  • Retransform: 
  -R0061
  -R0062
  -R0063
  -K091146
  • Inventory 
-Good Sequencing:
    o Backbone - C3RFP
    o pLux     - K091146
    o Lysis    - K112022
    o pLux     - R0063
    o pLux     - R1062
    o CI+RBS   - K081007
    o Cons.Prom- J23116
    o Cons.Prom- J23108
    o Pigment  - K274100
    o LuxI     - C0261
    o Lysis    - K112808
    o pLac     - R0010
    o LacI+RBS - J24679
  • To grow: 
   - K091146
   - R0063
   - J23108
   - J23116
  • Electrocompetent cells were made.


June 29

Gel Order: 

   - J23108
   - J23108 (Digest 2)
   - R0063  
   - R0063  (Digest 2)
   - K081007
   - K081007(Digest 2)
   - J23116 
   - J23116 (Digest 2)


6-29-10.jpg


  • Gel extract was done using Digest number 1. In this gel we ran the same part twice but from two different digests. Digest 1 was a 6 hour digest in the 37 degree Celsius water bath while Digest 2 was a 1 hour digest in the same water bath. As you can see, the results do not differ all that noticeably. That being established we went back to doing 1 hour digests.
  • The bands that were cut out are provided below: 
   - J23108
   - R0063
   - J23116
   - K081007

All these bands were purified.

June 30

  • On this gel, we ran the Colony PCR and the digests. The order is as follows:
  • Colony PCR: 
  - Ladder
  - A-J
  • Digests: 
  - Ladder
  - K091107 (digest 2)
  - K091107 
  - K091146 (digest 2)
  - K091146
  - J23116+J37033 (digest 2)
  - J23116+J37033
  - R0010 (digest 2)
  - R0010
6-30-10.jpg
  • The bands that were cut are marked with a square around them in the picture above. They were also then purified.
  • 3 bands that were cut: 
   - K091107
   - K091146
   - R0010


  • A second gel was run the same day. The order is provided below: 
  - Ladder
  - B1002
  - Lux R 2
  - C0261+J24679
6-30-10 001.jpg
  • As you can see, the only 2 bands that were cut were B1002 and C0261+J24679


  • Grow for tomorrow: 
  - K274100
  - K112022

July 1

  • Flasks minipreped: 
   - B1006
   - K274100
   - J23100
  • Digests: 
   - B1006 (EX)
   - K274100 (XP)
   - J23100 (SP)
  • Sent to the sequencing center: 
  1) B1002
  2) B1006
  3) S01414
  4) J23100
  5) C0261+J24679


  • Gel order: 
  - Ladder
  - B1006
  - K274100
  - J23100
7-1-10.jpg


  • All the bands appeared where we wanted them to so we cut out all three bands and then purified them.


  • Ligations:
VectorInserts
1)J23100K274100
2)J23100S01414
3)R0063a)K274100
b)C0261 + J24679
4)K091146a)K274100
b)C0261 + J24679
5)K091107a)K274100
b)C0261 + J24679
6)R0062a)K274100
b)C0261 + J24679
7)R0010K081007
  • Numbers 3-6 were each matched up with a) K274100 and b) C0261 + J24679. The rest of the vectors were matched with the insert directly to the right in the table.


  • Tomorrow: 
- Develop primers to figure out if K274100 and K112808 are correct.

July 2

  • Colony PCRs run on gel.The order is as follows: 
   - Ladder
   - 1A, 1B, 1C
   - 2A, 2B, 2C
   - 3A, 3B, 3C

   - Ladder
   - 4A, 4B, 4C
   - 5A, 5B, 5C
   - 6A, 6B, 6C
   - 7A, 7B, 7C
7-2-10.jpg


Unfortunately, we accidentally put ladder in all the Colony products. The picture is provided above. We could not use this information in any way other than to learn from our mistake.



Return to Notebook

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